Our long-term goal is to achieve real and detailed understanding of how yeast cells maintain balanced growth in the face of a fluctuating environment. Among the processes that have to be coordinated are the extraction of energy and metabolites from the environment; biosynthesis of appropriate amounts of hundreds of molecules, large and small; and the events of the cell division cycle, including replication of the DNA and assembly and segregation of subcellular organelles and structures. All of this coordination has to be done in such a way as to allow the cell to modify its activities, often on a very short time scale, when the environment changes. In the past few years, we have characterized the genome-wide gene expression changes associated with the maintenance of nutritional homeostasis. We developed technology that allows the identification, with high sensitivity and specificity, of single-nucleotide changes in the entire yeast genome and used it to examine the genomes of experimentally evolved strains. Our results have caused us to reassess the nature of the connections among survival, growth rate, stress response, cell cycle, and metabolic activity in yeast. We have now learned enough to formulate some specific and important hypotheses that we propose to test in the next grant period. Our specific aims are: 1) To test, by fluorescence in situ hybridization (FISH), the hypothesis that expression of many genes oscillates, in single cells growing slowly in unsynchronized cultures, in a pattern similar to that found in the yeast metabolic cycle seen in cultures whose oxidative metabolism has become synchronized in chemostats. If this test is positive, we will extend this finding to mammalian cells in culture using suitably chosen orthologs of oscillating yeast genes. (2) To test, by manipulating the expression of single genes in otherwise steady state cultures, the dynamic effect of changing the abundance of individual regulators. We will follow the effects of such manipulations of regulators implicated in glucose repression, heat shock, and cell cycle dynamics. (3) To systematically survey the genome for genes whose deletion or overproduction promotes survival during otherwise lethal starvation of auxotrophs and to test genes recovered from these screens for their ability to suppress glucose-wasting (Warburg Effect) during starvation. (4) To continue, using experimental evolution and direct screening, to characterize mutations that show increased fitness under conditions designed to challenge the ability of cells to maintain homeostasis, with an emphasis on conditions where respiration is required (i.e. with no fermentable sugar) or where fermentation is required (e.g. under anaerobic conditions or in strains lacking mitochondrial function). PUBLIC HEALTH RELEVANCE: It has long been established that the most fundamental activities required for cell growth and its control are carried out by genes and proteins whose sequence, function, and interactions are generally conserved among eukaryotes, from yeast to human. We study, in yeast, the mechanisms by which cells coordinate essential activities including (1) extraction of energy and metabolites from the environment; (2) orderly biosynthesis of varying amounts of a very large number of different molecules, large and small; (3) the events of the cell division cycle; and (4) assembly and segregation of a variety of subcellular organelles and structures. It is exactly these activities that fail to be coordinated in cancer cells, whose salient features include (1) inappropriate initiation of cell cycles; (2) failure to segregate chromosomes accurately; (3) characteristically aberrant patterns of gene expression (4) characteristic metabolic aberrations, such as the wasting of glucose through uncontrolled fermentation (Warburg Effect).